Project Summary Antimicrobial resistant bacterial infections account for over 23,000 deaths and over $20 billion per year in ex- cess healthcare costs in the USA. The development of new antimicrobials is an important strategy in the fight against antimicrobial resistance, but it is time consuming and expensive, and history has shown us that re- sistance to new agents develops within a few years of clinical use. Consequently, the prevention of antimicro- bial resistance is critically important to public health. Our interdisciplinary team proposes a preemptive strategy to characterize antimicrobial resistance before it arises using in vitro strategies that simulate clinical drug expo- sures. This proposal will focus on the recently approved long-acting lipoglycopeptides dalbavancin and orita- vancin, which have the longest half-lives of any available antibacterial (8-16 days). This slow clearance means that dalbavancin and oritavancin persist at low concentrations in the body for months after a single dose. These prolonged low-level exposures carry a high theoretical risk to select for resistance to the lipoglycopep- tides but also cross-resistance to related drugs, including vancomycin, the standard of care for treating the ?superbug? methicillin-resistant Staphylococcus aureus (MRSA). We have demonstrated that in vitro dalba- vancin exposure can select for resistance to vancomycin and daptomycin, and observed this cross-resistance emerge from a patient who received dalbavancin for an MRSA infection. Using a novel and comprehensive lipidomic approach, we observed for the first time that dalbavancin resistance was associated with a loss of phosphatidylglycerol and digalactosyldiacylglycerol species from the cell membrane. Using whole genome se- quencing, we identified mutations in 4 genes, one of which has been previously implicated in the emergence of vancomycin-nonsusceptible MRSA. We propose in Aim 1 to define the dalbavancin and oritavancin exposures that select for resistance and cross-resistance to the study antimicrobials (dalbavancin, oritavancin, vancomy- cin, and daptomycin). We hypothesize that the persistent low concentrations of dalbavancin and oritavancin after the last dose will readily select for resistance and cross-resistance among MRSA. We will test this hy- pothesis using pharmacokinetic/pharmacodynamic modeling techniques, which are well suited to this purpose and with which our team has considerable experience. In Aim 2, we will elucidate mechanisms of resistance to the study antimicrobials using a novel lipidomic approach, genome sequencing, and well-established biophysi- cal methods. We hypothesize that these changes in susceptibility will be concomitant with alterations in lipid expression, genetic variations associated with vancomycin nonsusceptibility, and other qualities such as cell wall thickening and reduced susceptibility to antimicrobial peptides produced by the immune system. This work is significant because understanding the risk of long-acting lipoglycopeptides to select for resistance is key to the development of policy for the optimal use of these agents and will elucidate lipoglycopeptide resistance mechanisms using a novel lipidomic strategy coupled with cutting edge genomic characterization.